Clear cell type renal cell carcinoma (CCRCC) is the most common and aggressive form of kidney cancer, and is among the most resistant of solid tumors to therapy. CCRCC is typically initiated by inactivation of the von Hippel Lindau (VHL) tumor suppressor gene, resulting in the constitutive activation of the hypoxia inducible factors, HIF-1? and HIF-2?. CCRCC progression is uniquely driven by HIF-2?, whereas HIF-1? plays a tumor suppressor role. Thus, HIF-2? is an attractive therapeutic target for CCRCC. Through a high throughput screening campaign, we have identified a series of compounds that selectively decrease HIF-2? protein and activity without affecting HIF-1?. We show that these compounds act by enhancing the binding of iron regulatory protein (IRP)-1 to the iron-responsive element (IRE) within the 5' untranslated region of HIF-2? mRNA, which inhibits HIF-2? translation. Using an unbiased global proteomic screen, we confirm Iron-sulfur Cluster Assembly 2 (ISCA2) as the molecular target of these compounds. ISCA2 regulates the incorporation of the iron-sulfur cluster into IRP-1, which modulates its IRE-binding activity. ISCA2 is non-transcriptionally induced by hypoxia, and is a putative pVHL target, suggesting that ISCA2 may play a hypoxia- or CCRCC- specific role. We observe that both ISCA2 and cellular iron are upregulated in CCRCC compared to paired normal kidney, and are significantly correlated. Inhibition of ISCA2 selectively decreases HIF-2? protein without affecting HIF-1?, and depletes cellular iron independently of HIF-2?. Significantly, ISCA2 inhibition by small molecules inhibits CCRCC xenograft growth, and decreases intra-tumoral HIF-2? protein. Thus, our hypothesis is that ?ISCA2 plays a central role in promoting the elevation of HIF-2? and cellular iron that drive CCRCC progression. Hence, the targeting of ISCA2 provides a novel strategy for the specific inhibition of HIF- 2? and depletion of cellular iron for the treatment of CCRCC?. Our first aim is to identify the mechanisms mediating ISCA2 induction in hypoxia; and to characterize its role in the regulation of HIF-2? and iron metabolism in CCRCC. Our second aim is to investigate the impact of ISCA2 modulation on CCRCC progression using in vitro and in vivo models, and to determine its physiological relevance using clinical samples of human CCRCC (samples from 600 patients obtained). Our third aim is to investigate the therapeutic impact of ISCA2 inhibition on HIF-2? and cytoplasmic iron, and to explore its use for the treatment of CCRCC. The overall goal of our studies is to identify the mechanisms by which ISCA2, HIF-2? and the deregulation of iron metabolism contribute to CRCC progression; and to determine whether ISCA2 inhibition will yield increased therapeutic benefit for patients with CCRCC.